This proposal focuses on the crystal structure determination of the human cannabinoid receptor hCB2, a G protein-coupled receptor (GPCR) that plays a central role in mediating the effects of cannabinoids and endocannabinoids in human physiology and pathology. An accurate structural understanding of the molecular mechanisms underlying GPCR activation and subsequent signal transmission to the cognate G proteins represents a major challenge for modern molecular biology and medicine. The proposed research aims at filling the knowledge gap on the structural basis of GPCR signaling by focusing on the crystallographic analysis of the hCB2 receptor in the active and inactive states. The specific aims are: 1) To determine the crystal structure of the hCB2 receptor in the active state. 2) To elucidate the structural basis for hCB2 receptor inactivation. To increase the stability and crystallizability of the hCB2 receptor, most of its intracellular loop 3 will be replaced with the T4 lysozyme (T4L) and the C-terminal cytoplasmic region of the receptor will be deleted. The optimized hCB2-T4L fusion protein will be expressed in insect cells and purified using affinity, ion exchange, and size exclusion chromatography. The purified receptor will be covalently complexed with a synthetic agonist or antagonist, and will be crystallized in lipidic mesophases. Complete diffraction data sets of the obtained crystals will be collected using synchrotron radiation and the structures will be determined using cutting-edge macromolecular crystallography methods. The successful completion of these studies will have a major impact on biomedical research and human health. The obtained structural knowledge will greatly facilitate the development of hCB2-selective agonists, partial agonists, and antagonists. Because of the central role of the hCB2 receptor in mediating endocannabinoid signaling, new treatments will be developed for a number of medical conditions, including inflammatory and neuropathic pain, hepatic fibrosis, atherosclerosis, and osteoporosis. PUBLIC HEALTH RELEVANCE: The human cannabinoid receptor hCB2 plays a central role in mediating the (endo)cannabinoid signaling in human physiology and pathology. The proposed research focuses on the elucidation of the mechanisms underlying hCB2 function at the atomic level using X-ray crystallography. Accurate knowledge of the three- dimensional structure of hCB2 will advance the research in the (endo)cannabinoid and GPCR fields, and will effectively facilitate the development of innovative therapeutics for serious human diseases, including drugs of abuse and HIV.